Components of the outermost surface layer of the B. anthracis spore, called the exosporium, are first to come in contact with the host, and their interactions with host defenses are likely to have an important influence on the fate of ingested spores. A major component of the exosporium is a glycoprotein called BclA. It contains about 70 PTG tripeptide repeats, several of which possess tetrasaccharide side chains composed of a unique substituted amino sugar and three rhamnose residues. The role(s) of BclA in anthrax pathogenesis has not been established but other rhamnose-containing glycoeonjugates have been found to possess a variety of potent biological activities, including activation of macrophages and inhibition of neutrophil phagocytosis. The first specific aim of this proposal is to identify the precise positions in the protein backbone of BclA that are glycosylated and also to determine the extent of substitution at the various sites. This task is complicated by the fact that the amino acid sequence of BclA is not amenable to the preparation of glycopeptide fragments and novel molecular genetic approaches will be used to locate the glycosylation sites. The second specific aim is to identify the genes involved in the assembly of the rhamnose-containing tetrasaccharides of BclA. These genes will be identified by systematically mutationally inactivating candidate genes selected on the basis of either their putative assignment in the B. anthracis genome or their similarity to known genes in other bacterial species. The effects of mutating these genes on spore morphology, viability, germination, infectivity, and virulence will be determined. The effects of the mutations on oligosaccharide structure will be studied using a variety of chemical and physical methods, including mass spectrometry and NMR methods. The third specific aim is to prepare and evaluate a novel oligosaccharide-protein conjugate vaccine in which the tetrasaccharide side chains of BclA are covalently linked to recombinant protective antigen of B. anthracis. Antibodies elicited by the vaccine should promote the phagocytosis and killing of anthrax spores as well as protect against toxemia. In one group of experiments the vaccine will be administered to mice subcutaneously using an aluminum hydroxide adjuvant. In other experiments it will be administered intranasally with a cholera toxin adjuvant in order to elicit a mucosal immune response. In both cases the ability of the vaccines to protect a susceptible strain of mice from a lethal inhalational anthrax infection will be assessed.